Gabriele Rincke

Glycogen synthase kinase-3 interacts with and phosphorylates estrogen receptor alpha and is involved in the regulation of receptor activity.

Like other steroid hormone receptors, estrogen receptor-α (ERα) is a substrate for protein kinases, and phosphorylation has profound effects on the function and activity of this receptor. A number of different kinases have been implicated in ERα regulation. In this report we show by mutational analysis and in vitro kinase assays that ERα is a substrate for glycogen synthase kinase-3 (GSK-3) in vitro and is phosphorylated on two sites, the Ser-102, -104, and -106 motif and Ser-118, both located in the N-terminal transcription activation function (AF-1) domain. GSK-3 forms a complex with ERα in vivo as demonstrated by co-immunoprecipitation from cell lysates. The GSK-3 inhibitor lithium chloride was used to determine the role of GSK-3 in phosphorylation of Ser-102, -104, and -106 and Ser-118 in vivo and to explore the role of these serines in the regulation of ERα function. Treatment of cells with lithium chloride resulted in dephosphorylation of Ser-104 and -106 and Ser-118, which suggests these serine residues as targets for GSK-3 in vivo. Our results further suggest that ERα phosphorylation by GSK-3 stabilizes ERα under resting conditions and modulates ERα transcriptional activity upon ligand binding. Inhibition and constitutive activation of GSK-3, both, resulted in inhibition of ERα transcriptional activity, indicating a function of active as well as inactive GSK-3 in ERα regulation. These findings uncover a novel mechanism for the regulation of ERα-mediated estrogen signaling controlled by a dual action of GSK-3.

Phosphorylation of Protein Kinase Cδ (PKCδ) at Threonine 505 Is Not a Prerequisite for Enzymatic Activity EXPRESSION OF RAT PKCδ AND AN ALANINE 505 MUTANT IN BACTERIA IN A FUNCTIONAL FORM

A structural feature shared by many protein kinases is the requirement for phosphorylation of threonine or tyrosine in the so-called activation loop for full enzyme activity. Previous studies by several groups have indicated that the isotypes α, βI, and βII of protein kinase C (PKC) are synthesized as inactive precursors and require phosphorylation by a putative “PKC kinase” for permissive activation. Expression of PKCα in bacteria resulted in a nonfunctional enzyme, apparently due to lack of this kinase. The phosphorylation sites for the PKC kinase in the activation loop of PKCα and PKCβII could be identified as Thr497 and Thr500, respectively. We report here that PKCδ, contrary to PKCα, can be expressed in bacteria in a functional form. The activity of the recombinant enzyme regarding substrate phosphorylation, autophosphorylation, and dependence on activation by 12-O-tetradecanoylphorbol-13-acetate as well as the Km values for two substrates are comparable to those of recombinant PKCδ expressed in baculovirus-infected insect cells. By site-directed mutagenesis we were able to show that Thr505, corresponding to Thr497 and Thr500 of PKCα and PKCβII, respectively, is not essential for obtaining a catalytically competent conformation of PKCδ. The mutant Ala505 can be activated and does not differ from the wild type regarding activity and several other features. Ser504 can not take over the role of Thr505 and is not prerequisite for the kinase to become activated, as proven by the unaffected enzyme activity of respective mutants (Ala504 and Ala504/Ala505). These results indicate that phosphorylation of Thr505 is not required for the formation of functional PKCδ and that at least this PKC isoenzyme differs from the isotypes α, βI, and βII regarding the permissive activation by a PKC kinase.

Requirements of protein kinase cdelta for catalytic function. Role of glutamic acid 500 and autophosphorylation on serine 643.

Recently, we reported that, in contrast to protein kinase C (PKC)α and βII, PKCδ does not require phosphorylation of a specific threonine (Thr505) in the activation loop for catalytic competence (Stempka et al. (1997) J. Biol. Chem. 272, 6805–6811). Here, we show that the acidic residue glutamic acid 500 (Glu500) in the activation loop is important for the catalytic function of PKCδ. A Glu500 to valine mutant shows 76 and 73% reduced kinase activity toward autophosphorylation and substrate phosphorylation, respectively. With regard to thermal stability and inhibition by the inhibitors Gö6976 and Gö6983 the mutant does not differ from the wild type, indicating that the general conformation of the molecule is not altered by the site-directed mutagenesis. Thus, Glu500 in the activation loop of PKCδ might take over at least part of the role of the phosphate groups on Thr497and Thr500 of PKCα and βII, respectively. Accordingly, PKCδ exhibits kinase activity and is able to autophosphorylate probably without posttranslational modification. Autophosphorylation of PKCδ in vitro occurs on Ser643, as demonstrated by matrix-assisted laser desorption ionization mass spectrometry of tryptic peptides of autophosphorylated PKCδ wild type and mutants. A peptide containing this site is phosphorylated also in vivo, i.e. in recombinant PKCδ purified from baculovirus-infected insect cells. A Ser643 to alanine mutation indicates that autophosphorylation of Ser643 is not essential for the kinase activity of PKCδ. Probably additional (auto)phosphorylation site(s) exist that have not yet been identified.

DIK, a Novel Protein Kinase That Interacts with Protein Kinase Cδ CLONING, CHARACTERIZATION, AND GENE ANALYSIS

A novel serine/threonine kinase, termed DIK, was cloned using the yeast two-hybrid system to screen a cDNA library from the human keratinocyte cell line HaCaT with the catalytic domain of rat protein kinase Cδ (PKCδcat) cDNA as bait. The predicted 784-amino acid polypeptide with a calculated molecular mass of 86 kDa contains a catalytic kinase domain and a putative regulatory domain with ankyrin-like repeats and a nuclear localization signal. Expression of DIK at the mRNA and protein level could be demonstrated in several cell lines. Thedik gene is located on chromosome 21q22.3 and possesses 8 exons and 7 introns. DIK was synthesized in an in vitro transcription/translation system and expressed as recombinant protein in bacteria, HEK, COS-7, and baculovirus-infected insect cells. In the in vitro system and in cells, but not in bacteria, various post-translationally modified forms of DIK were produced. DIK was shown to exhibit protein kinase activity toward autophosphorylation and substrate phosphorylation. The interaction of PKCδcat and PKCδ with DIK was confirmed by coimmunoprecipitation of the proteins from HEK cells transiently transfected with PKCδcat or PKCδ and DIK expression constructs.

Proteolytic cleavage of protein kinase Cμ upon induction of apoptosis in U937 cells

Treatment of U937 cells with various apoptosis‐inducing agents, such as TNFα and β‐D‐arabinofuranosylcytosine (ara‐C) alone or in combination with the phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), bryostatin 1 or cycloheximide, causes proteolytic cleavage of protein kinase Cμ (PKCμ) between the regulatory and catalytic domain, generating a 62 kDa catalytic fragment of the kinase. The formation of this fragment is effectively suppressed by the caspase‐3 inhibitor Z‐DEVD‐FMK. In accordance with these in vivo data, treatment of recombinant PKCμ with caspase‐3 in vitro results also in the generation of a 62 kDa fragment (p62). Treatment of several aspartic acid to alanine mutants of PKCμ with caspase‐3 resulted in an unexpected finding. PKCμ is not cleaved at one of the typical cleavage sites containing the motif DXXD but at the atypical site CQND378/S379. The respective fragment (amino acids 379–912) was expressed in bacteria as a GST fusion protein (GST‐p62) and partially purified. In contrast to the intact kinase, the fragment does not respond to the activating cofactors TPA and phosphatidylserine and is thus unable to phosphorylate substrates effectively.

The induction of ornithine decarboxylase by the tumor promoter TPA is controlled at the post-transcriptional level in murine Swiss 3T3 fibroblasts.

The expression of ornithine decarboxylase (ODC) mRNA after treatment of murine Swiss 3T3 cells with the tumor promoter TPA was studied. The induction of ODC mRNA was detectable after 20-40 min, peaked after 60-120 min and declined within 24 hrs. Using an in vitro nuclear transcription assay, we found that the polymerase II density on the ODC gene is not affected by TPA treatment. Additionally, we were able to detect stable ODC mRNAs in cycloheximide pretreated fibroblasts. These two different experimental approaches lead us to the interpretation that in Swiss 3T3 cells TPA controls ODC expression predominantly at the post transcriptional level by prolonging the half-life of ODC-mRNA.

cDNA-cloning, sequencing and expression in glucocorticoid-stimulated quiescent Swiss 3T3 fibroblasts of mouse lipocortin I.

We isolated and sequenced mouse lipocortin I cDNA clones from a lambda gt10 cDNA library prepared from Swiss 3T3 mRNA. The homology with human lipocortin I at the amino acid level is 86%. When confluent layers of Swiss 3T3 cells were stimulated with 10% fetal calf serum, expression of lipocortin I was strongly stimulated. In parallel, DNA synthesis was induced with a peak at 24 hours after glucocorticoid treatment indicating induction of cell proliferation. In the absence of serum glucocorticoid treatment provoked neither induction of DNA synthesis nor expression of lipocortin I. We conclude that serum contains an unidentified factor, which acts synergistically with glucocorticoids on cell proliferation and lipocortin I expression.

Immunological demonstrations of εPKC Murine tissue distribution, ontogeny, cellular localization and translocation

An antiserum raised against an εPKC‐specific peptide recognizes εPKC with an apparent molecular weight of 97 kDa in cytosol of mouse brain. No cross‐reaction with α,β,γPKC or the δPKC‐like p76‐kinase is observed. εPKC is mainly present in brain. Just traces of this PKC isoenzyme can be detected in some other murine tissues. Ontogenetic studies indicate that the amount of εPKC in murine brain increases constantly and reaches a maximal level at day 7 after birth. Upon TPA activation εPKC is translocated from the cytosol to the particulate fraction in a brain homogenate.

Inhibition of the estrogen-induced synthesis of vitellogenin mRNA in chick liver by tamoxifen

Abstract Cloned vitellogenin cDNA (labelled with 32 P) was used as a probe for measuring vitellogenin mRNA sequences in RNA preparations from the liver of chicks treated with estradiol and/or tamoxifen. For the first time it was shown that the antiestrogen tamoxifen inhibits the estradiol-induced synthesis of vitellogenin mRNA in chick liver. This inhibition correlates very well with a reduced capacity of the liver to synthesize vitellogenin. Furthermore, evidence is presented that tamoxifen lacks any agonistic activity in chick liver. Vitellogenin mRNA is not measurable after tamoxifen alone.